Pregnant women are at high risk for invasive Salmonella infection, which can cause both maternal and fetal complications. We found that Salmonella infection in pregnant mice caused rapid fetal and maternal death due to massive bacterial proliferation in placental trophoblast cells (TBCs). In vitro, Salmonella flourished in human trophoblast-derived choriocarcinoma cells. However, there is a gap in our knowledge regarding the mechanism of increased susceptibility to Salmonella infection during pregnancy. Our long-term objective is to develop strategies that can control the outcome of intracellular infections in pregnant women. The overall objective of this application is to identify the molecular mechanisms that render TBCs highly susceptible to Salmonella. Our central hypothesis is that placental TBCs are productively infected by Salmonella and provide a unique intra- cellular niche that permits uncontrolled virulent Salmonella replication due to an ineffectie immune response, resulting in placental death. Our central hypothesis will be tested by three Specific Aims which will identify: 1) the mechanism of uncontrolled intracellular growth of Salmonella in TBCs, 2) the role(s) of IFN-?? in susceptibility of TBCs to Salmonella, and 3) the mechanism of Salmonella-induced placental inflammation. Studies will be performed in vitro using primary isolated human trophoblast cells and placental explants, and in vivo using pregnant mouse models. Specific Aim 1 will test the hypothesis that Salmonella thrive within a unique intracellular environment of trophoblast cells. We will elucidate whether different trophoblast subpopulations of differing gestational age specifically promote Salmonella replication due to specific cell-cell interactions. We will also identify the role of bacterial injectisome virulence factors in facilitating entry into TBCs and the relative role of trophoblast phagocytosis. Lastly, we will characterize the molecular features of the intracellular environment (sub-cellular vacuole) in which Salmonella proliferates. Specific Aim 2 will test the hypothesis that trophoblastic hypo-responsiveness to IFN-?? contributes to susceptibility to Salmonella. We will test whether IFN-? pre- treatment of TBCs can contain Salmonella infection, how Salmonella modulates the JAK-STAT-1 signaling pathway in TBCs, and if knocking down negative-feedback inhibitors of IFN-?? signaling such as SOCS expression can aid resolution of TBC-Salmonella infection. Specific Aim 3 will test the working hypothesis that the mechanism of ST-induced TBC death is distinct from that encountered in other infected cells and contributes to overt inflammation. We will identify differential inflammation (IL-1?, IL-18, caspase) and/or cell death signaling (apoptosis, pyroptosis or necroptosis) as a cause of TBC susceptibility to Salmonella. Overall we expect to identify the relative importance of unique trophoblast features versus Salmonella virulence factors in destruction of the placenta. This contribution will significantly reshape our understanding of infection risk during pregnancy. Successful completion of this study will represent the critical first step in the continuum of research requird to develop rational treatment regimens for management of placental infections during pregnancy. PUBLIC HEALTH RELEVANCE: Pregnant women have an increased susceptibility to infection by food-borne pathogens such as Salmonella, and these infections are associated with severe complications of pregnancy, including miscarriage, premature and still birth, and chorioamnionitis. These studies will provide important insights into the unique features of placental trophoblast cells and Salmonella virulence factors that lead to increased pathogenicity in pregnant women.